The Selective Nav1.7 Inhibitor, PF-05089771, Interacts Equivalently with Fast and Slow Inactivated Nav1.7 Channels.

Voltage-gated sodium (Nav) channel inhibitors are used clinically as analgesics and local anesthetics. However, the absence of Nav channel isoform selectivity of current treatment options can result in adverse cardiac and central nervous system side effects, limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Nav1.7 sodium channels in the sensation of pain, thus making this channel an attractive target for new pain therapies. We previously identified a novel, state-dependent human Nav1.7 selective inhibitor (PF-05089771, IC50 = 11 nM) that interacts with the voltage-sensor domain (VSD) of domain IV. We further characterized the state-dependent interaction of PF-05089771 by systematically varying the voltage, frequency, and duration of conditioning prepulses to provide access to closed, open, and fast- or slow-inactivated states. The current study demonstrates that PF-05089771 exhibits a slow onset of block that is depolarization and concentration dependent, with a similarly slow recovery from block. Furthermore, the onset of block by PF-05089771 develops with similar rates using protocols that bias channels into predominantly fast- or slow-inactivated states, suggesting that channel inhibition is less dependent on the availability of a particular inactivated state than the relative time that the channel is depolarized. Taken together, the inhibitory profile of PF-05089771 suggests that a conformational change in the domain IV VSD after depolarization is necessary and sufficient to reveal a high-affinity binding site with which PF-05089771 interacts, stabilizing the channel in a nonconducting conformation from which recovery is slow.

Differential regulation of CaV1.2 channels by cAMP-dependent protein kinase bound to A-kinase anchoring proteins 15 and 79/150.

The CaV1.1 and CaV1.2 voltage-gated calcium channels initiate excitation-contraction coupling in skeletal and cardiac myocytes, excitation-transcription coupling in neurons, and many other cellular processes. Up-regulation of their activity by the ß-adrenergic-PKA signaling pathway increases these physiological responses. PKA up-regulation of CaV1.2 activity can be reconstituted in a transfected cell system expressing CaV1.2Δ1800 truncated at the in vivo proteolytic processing site, the distal C-terminal domain (DCT; CaV1.2[1801-2122]), the auxiliary α2δ and ß subunits of CaV1.2 channels, and A-kinase anchoring protein-15 (AKAP15), which binds to a site in the DCT. AKAP79/150 binds to the same site in the DCT as AKAP15. Here we report that AKAP79 is ineffective in supporting up-regulation of CaV1.2 channel activity by PKA, even though it binds to the same site in the DCT and inhibits the up-regulation of CaV1.2 channel activity supported by AKAP15. Mutation of the calcineurin-binding site in AKAP79 (AKAP79ΔPIX) allows it to support PKA-dependent up-regulation of CaV1.2 channel activity, suggesting that calcineurin bound to AKAP79 rapidly dephosphorylates CaV1.2 channels, thereby preventing their regulation by PKA. Both AKAP15 and AKAP79ΔPIX exert their regulatory effects on CaV1.2 channels in transfected cells by interaction with the modified leucine zipper motif in the DCT. Our results introduce an unexpected mode of differential regulation by AKAPs, in which binding of different AKAPs at a single site can competitively confer differential regulatory effects on the target protein by their association with different signaling proteins.

Molecular mechanism of calcium channel regulation in the fight-or-flight response.

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by Ca(V)1 channels through activation of ß-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3',5'-monophosphate-dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac Ca(V)1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of Ca(V)1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α2δ1 and ß(2b) subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca(V)1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal Ca(V)1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for Ca(V)1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.

Isolation and characterization of a high affinity peptide inhibitor of ClC-2 chloride channels.

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K(D) of approximately 20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.

State-dependent inhibition of cystic fibrosis transmembrane conductance regulator chloride channels by a novel peptide toxin.

Peptide toxins from animal venom have been used for many years for the identification and study of cation-permeable ion channels. However, no peptide toxins have been identified that interact with known anion-selective channels, including cystic fibrosis transmembrane conductance regulator (CFTR), the protein defective in cystic fibrosis and a member of the ABC transporter superfamily. Here, we describe the identification and initial characterization of a novel 3.7-kDa peptide toxin, GaTx1, which is a potent and reversible inhibitor of CFTR, acting from the cytoplasmic side of the membrane. Thus, GaTx1 is the first peptide toxin identified that inhibits a chloride channel of known molecular identity. GaTx1 exhibited high specificity, showing no effect on a panel of nine transport proteins, including Cl(-) and K(+) channels, and ABC transporters. GaTx1-mediated inhibition of CFTR channel activity is strongly state-dependent; both potency and efficacy are reduced under conditions of elevated [ATP], suggesting that GaTx1 may function as a non-competitive inhibitor of ATP-dependent channel gating. This tool will allow the application of new quantitative approaches to study CFTR structure and function, particularly with respect to the conformational changes that underlie transitions between open and closed states.

The block of CFTR by scorpion venom is state-dependent.

Cystic fibrosis transmembrane conductance regulator (CFTR) adenosine triphosphate-dependent chloride channels are expressed in epithelial cells and are associated with a number of genetic disorders, including cystic fibrosis. Venom of the scorpion Leirus quinquestriatus hebraeus reversibly inhibits CFTR when applied to its cytoplasmic surface. To examine the state-dependence of inhibition we recorded wild-type and mutant CFTR channel currents using inside-out membrane patches from Xenopus oocytes. Application of either venom or diphenylamine-2-carboxylate to channels that were either activated (open) or resting (closed) indicate primarily closed state-dependent inhibition of CFTR by venom, whereas diphenylamine-2-carboxylate showed no state-dependence of block. Efficacy of venom-mediated macroscopic current inhibition was inversely related to channel activity. Analysis of single-channel and macropatch data indicated that venom could either inhibit channel opening, if it binds during an interburst closed state or in the absence of cytosolic adenosine triphosphate, or introduce new intraburst closed states, if it binds during an open event. The on-rate of venom binding for intraburst block could be modulated by changing CFTR activity with vanadate or adenylyl-imidodiphosphate, or by introducing the Walker A mutation K1250A. These findings represent the first description of state-dependent inhibition of CFTR and suggest that the active toxin could be used as a tool to study the conformational changes that occur during CFTR gating.

Structure elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway.

The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcbeta1Cer --> Manbeta4Glcbeta1Cer [MacCer] --> GlcNAcbeta3Manbeta4Glcbeta1Cer [At(3)Cer] --> GalNAcbeta4- GlcNAcbeta3Manbeta4Glcbeta1Cer [At(4)Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galbeta3Manbeta4Glcbeta1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAcalpha4Galbeta3Manbeta4- Glcbeta1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAcalpha4 and Galbeta3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcbeta4 in the arthro-series penta- and hexaglycosylceramide structures (At(5)Cer and At(6)Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAcalpha4 and Galbeta3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galbeta3-T capable of using Manbeta4Glcbeta1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.

CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore.

We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.

Inhibition of CFTR channels by a peptide toxin of scorpion venom.

Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified that interact with known anion-selective channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR channels are expressed in epithelial cells and are associated with several genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the Cl- permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered by the lack of a high-affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion Leiurus quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing single-channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or adenosine 5'-(beta,gamma-imido)triphosphate, and block was strengthened on reduction of extracellular Cl- concentration, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore-block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function.